Synthetic oligonucleotides (oligodeoxyribo- and oligoribonucleotide) have become indispensable tools in modern biological sciences. The protocols for chemical synthesis and modifications of these molecules have been simplified to an extent that even a non-chemist, the actual user can synthesize these molecules without much difficulties. However, the support functionalization has been one of the time consuming tasks. Moreover, one still requires to use at least eight different (four for oligodeoxyribo- and four for oligoribonucleotides) supports, each bearing a separate nucleoside corresponding to the 3'-terminus of the desired oligomer. A number of base labile group containing phosphoramidite synthons have been proposed and used (shown in sets II-IV) to speed up the post synthesis (deprotection) time. Therefore, one would require to prepare the pre-functionalized supports accordingly and hence the number of pre-derivatized supports would be very large. This number may even be more when 3'-terminus of the desired oligomer contains nucleotide other than the normal one. In the last few years, some attempts have been made to develop universal supports to eliminate the use of at least eight different supports. Gough et al. (Tet. Lett. 24, 1983, 5321) have proposed the use of a universal support that consisted of controlled pore glass derivatized with 2'(3')-O-benzoyluridine-5'-O-succinyl residue and demonstrated its usefulness for the synthesis of oligodeoxyribo- and oligoribonucleotides via phosphotriester or the phosphite approach. The support contains a nucleosidic material which does not get incorporated in the oligonucleotide chain and goes waste. Further, the functionalization of the support involves multi steps (4 steps) synthesis and purification procedure. The cleaving of oligodeoxyribo- and oligoribonucleotides was effected by the use of aq. ammonia for 24 h at 60-65.degree. C. followed by treatment with lead cation for 18 h and aq. ammonia-pyridine (2:1) for 16 h at 50.degree. C., respectively, in the sealed vials. In a more recent publication, Gough and co-workers (Tet. Lett., 36, 1995, 27) have further modified the functionalization of the universal support by the use of an adaptor, 2'(3')-O-benzoyluridine-5'-O-(cyanoethyl-N,N-diisopropyl-phosphoramidite) and coupled it to a standard T-support, following phosphoramidite chemistry. The functionalized support was subsequently used for the synthesis of oligonucleotides. The deprotection of oligodeoxyribo- and oligoribonucleotides was achieved by the treatment of the support with 25 ml aq. ammonia for 48 h at 60.degree. C. and 25 ml aq. ammonia-pyridine mixture (4:1, v/v) for 24 h at 50.degree. C., respectively, in a pressure vial. However, the universal support has still the following serious limitations. The support contains two nucleosidic units instead of one in earlier support (very expensive material) which do not get incorporated in the oligonucleotide chain and go waste. The adaptor used for the functionalization of universal support involves multi step synthesis and purification strategy and the yield of the final product is low, making the support functionalization a time consuming (2-3d), tedious and expensive. Since the deprotection time of oligodeoxyribo- and oligoribonucleotides synthesized using conventional or base labile protecting group containing nucleosidephosphoramidite synthons is very less e.g. 20 min in aq. ammonia at 60.degree. C. or 1-2 h in aq. ammonia at room temperature or 5 min in aq. ammonia-methylamine at 65.degree. C. or 1-2 h in aq. ammoniamethylamine at room temperature, these requirements are not met by the universal supports proposed by Gough et al.(Tet. Lett., 24, 1983, 5321; ibid. 36, 1995, 27).
In order to demonstrate the use of a non-nucleosidic linker molecule for the preparation of universal support, the inventors of application WO 95/01987 have employed a commercially available reagent, 3-glycidyloxypropyltrimethoxysilane, for the preparation of an epoxide based support. The principle is based on the opening up of the epoxide ring in the synthesizer itself by treatment with halogenated acid which generates a secondary hydroxyl group for the chain elongation and primary hydroxyl in the protected form. The cleaving of the oligomer chain from the support and removal of protecting groups from nucleic bases have been achieved by aq. ammonia treatment at 100.degree. C. for 1 h. In examples 1-7, they have demonstrated the use of the same epoxide based variuos supports for the synthesis of DNA and RNA while in example 8, glycol type of support has been generated but restricted to primary hydroxyl groups (on adjacent carbon atoms). The deprotection conditions employed in this report are not compatible with the established deprotection conditions particularly with RNA and base labile oligonucleotides.
Recently, one more universal support (Nucl. Acids Res., 1996, 24, 2793) containing a 1-O-(4,4'- dimethoxytrityl)-2-O-succinyl-3-N-allyloxycarbonylpropane immobilized on aminopropyl-CPG has been proposed. However, the release of oligomers from the support involves three steps process.
Therefore, there is a need to develop a universal polymer support suitable for the synthesis of oligodeoxyribo- and oligoribonucleotides and compatible to the existing methods of their synthesis and deprotection using nucleosidephosphoramidite synthons carrying conventional or base labile protecting groups for exocyclic amino functionalities.
The main objective of the present invention is, therefore, to provide a process for the preparation of an improved universal polymer support useful for the synthesis of all possible oligodeoxyribo- and oligoribonucleotides.
Another objective of the present invention is to provide a process for the preparation of an improved universal polymer support devoid of any nucleosidic material, suitable for the synthesis of all possible oligodeoxyribo- and oligoribonucleotides.
Still another objective of the present invention is to provide a process for the preparation of an improved universal polymer support compatible to the existing methods of oligonucleotide synthesis and deprotection using nucleosidephosphoramidite synthons carrying conventional or base labile protecting groups for exocyclic amino functionalities.
Yet another objective of the present invention is to provide a process for the preparation of oligonucleotides using an improved universal polymer support compatible to the existing methodology of synthesis of oligonucleotides employing base labile group containing nucleosidephosphoramidite synthons in automated DNA synthesizers and simultaneous cleaving of oligonucleotide chains from the support and removal of protecting groups from exocyclic amino functionalities of nucleic bases and 2-cyanoethyl from internucleotidic phosphate functions of all possible oligonucleotides under microwave irradiation.
Still another objective of the present invention is to provide a process for the cleaving of oligonucleotides from an improved universal polymer support and simultaneous removal of protecting groups, labile or conventional, from the exocyclic amino functionalities of nucleic bases and 2-cyaneothyl from the internucleotidic phosphate functions of all possible oligonucleotides (oligodeoxyribo- and oligoribo-) under established deprotection conditions (20 min at 60.degree. C. in aq. ammonia or 60-120 min at room temperature in aq. ammonia or 75-90 min at room temperature in aq. ammonia-methylamine mixture or 5 min at 65.degree. C. in aq. ammonia-methylamine).
Based on the above objectives, we designed some supports of the structures shown below: ##STR3##
The supports I to IV were found suitable for the synthesis of all possible oligodeoxyribo- and oligoribonucleotides using nucleosidephosphoramidite synthons carrying conventional and base labile protecting groups for exocyclic amino functionalities as shown in sets I-IV, but the oligonucleotides did not cleave to give free 3-hydroxyl groups from the support I (based on two primary hydroxyl groups on adjacent carbon atoms) with aq. ammonia in 24 h at 60.degree. C. and hence not suitable for oligonucleotide synthesis. In case of supports II and III (based on one primary and one secondary hydroxyl groups on adjacent carbons), same problem was encountered under normal deprotection conditions (discussed in objectives) while the cleavage from the support II has been shown by the inventors of application WO 95/01987 with aq. ammonia at very high temperature (100.degree. C., 1 h) which is totally undesirable in oligonucleotide synthesis particularly in case of RNA and base sensitive oligonucleotides. However, the support IV was found to be compatible with the normal deprotection conditions. 3'-Hydroxyl group containing oligomers were obtained under all possible fast deprotection conditions for DNA, RNA and base sensitive oligomers (Table 1).
In search of a suitable mechanism for the rapid cleavage of oligonucleotides under mild conditions, we noticed that Gough and co-workers had already reported that a nucleotide having 3'-terminal protection with a ribonucleoside linked by its 2'- or 3'-hydroxyl group to a 3'-phosphate is removed as its 2',3'-cyclic phosphate by lead cation. In this process a nucleotide or oligonucleotide gets removed with 3'-hydroxyl function. This concept led Gough and co-workers to develop two universal
TABLE 1 __________________________________________________________________________ Comparison of nucleosidic and non-nucleosidic universal supports available with the proposed universal support Universal support with Cleavage % nucleosidic material conditions Cleavage __________________________________________________________________________ 1 #STR4## Aq.NH3, 24 h, 60.degree. C. +Pb(OAc)2, 18 h, r.t. (Not compatible to base labile pr. groups) 100% 2 #STR5## Aq.NH3 (25 ml), 48h 65.degree. C. (Not compatible to conventional or base labile pr. groups) 100% Universal supports with non-nucleosidic material 3 #STR6## Aq.NH3 + 0.5M LiCl, 55.degree. C., 5 h (not comapatible to base labile pr. 100%ps) Pd(0), 50.degree. C., 15 min +0.1NTEAA+aq.NH3, 2 h 100% 4 #STR7## +Aq.NH3, 55.degree. C., 5 h (Not compatible to base labile pr. groups) ##STR8## 5 Aq.NH3, 100.degree. C., 1 h (Undesirable conditions for DNA & RNA) 90% 6 #STR9## Aq.NH3, 60.degree. C., 20 min or aq.NH3 r.t., 1-2h (Compatible to pr. groups of Set II-IV) 100% - 6 #Aq.NH3 + CH3NH2, 65.degree. C., 5 min/ Ag.NH3 + CH3NH2, r.t., 90 min/ Aq.NH3 + CH3NH2, r.t., 75, min Compatible to pr.groups of Set __________________________________________________________________________ I-IV) 100%
supports (Gough et al., Tet. Lett., 24, 1983, 5321; ibid. 36, 1995, 27) useful for the preparation of oligonucleotides. Because of the rigid rotation of the hydroxyl groups in uridine, oligodeoxyribo- and oligoribonucleotides take considerable time (48 h) for cleaving from the universal supports. Similarly the supports prepared using non-nucleosidic linker e.g. using polymer supported 1,4-anhydroerythritol also not compatible to the deprotection conditions required for the cleavage of oligonucleotides synthesized using base labile nucleosidephosphoramidite synthons. From all of these studies, one of the most important criteria i.e. the selection of the organic moiety for the preparation of universal support is clear that the selected moiety should contain at least two secondary hydroxyl groups on the adjacent carbon atoms. As far as hindered rotation of hydroxyl groups in the ring structure is concerned, this might be taken care of if the source of cis diol groups (both secondary) is replaced by an organic molecule having a pair of cis hydroxyl groups (both secondary) with free rotation. The free rotation of vicinal hydroxyl groups will help in rapid formation of cyclic phosphate under mild alkaline conditions and hence will lead to rapid deprotection of oligodeoxyribo- and oligoribonucleotides from an improved universal polymer support.
Process details
Accordingly, the present invention provides an improved process for the preparation of a universal polymer support of the structure provided with the specifications, devoid of any nucleosidic material useful for the synthesis of oligodeoxyribo- and oligoribonucleotides. The process also relates to the cleaving of oligonucleotides (both oligodeoxyribo- and oligoribo-) from the support and simultaneous removal of protecting groups, labile or conventional from the exocyclic amino functionalities of nucleic bases and 2-cyanoethyl from internucleotidic phosphate functions of all possible oligodeoxyribo- and oligoribonucleotides, which comprises:
(i) treating an organic aliphatic molecule having a pair of hydroxyl groups (both are at secondary position) at adjacent carbon atoms with 4,4'-dimethoxytrityl chloride and isolating the monosubstituted substance ##STR11## where R.dbd.H--(CH.sub.2).sub.n -- and R'=--.sub.2 --OH, --(CH.sub.2).sub.n --H; n=1-4; DMTr=4,4'-dimethoxytrityl.
(ii) treating the monosubstituted substance obtained in step (i) with one equivalent of a homobifunctional alkanoic acid halide and transferring the mixture to a polymer support carrying hydroxyl or aminoalkyl functionalities.
(iii) treating the polymer support obtained in step (ii) with dry alkanol for blocking the residual functional groups (capping) on the universal polymer support followed by washing with dry alkanol and dialkyl ether, respectively.
The organic aliphatic molecule having a pair of hydroxyl groups (both are at secondary positions) on adjacent carbons used for making monosubstituted tritylated derivative may be selected from butane-2,3-diol, 1,2,3-trihydroxyheptane, 1,2,3-hexanetriol and the like.
The homobifunctional alkanoic acid halide used in step (ii) may be selected from oxalyl chloride, succinoyl chloride, adipoyl chloride and the like.
The polymer support employed in step (ii) may be selected from organic or inorganic polymers e.g. controlled pore glass (CPG) with variable pore size and linker arm, silica gel (porous or non-porous), cross-linked polystyrene having hydroxyl or aminoalkyl functions and the like.
The alkanol used in step (iii) for capping purpose may be selected from methanol, ethanol, propanol and the like.
The improved universal polymer support was then employed for the synthesis of oligonucleotides which comprises:
(i) synthesizing oligodeoxyribo- and oligoribonucleotides on an improved universal polymer support following phosphoramidite chemistry employing conventional or labile protecting groups for nucleic bases.
(ii) treating the oligodeoxynucleotide and oligoribonucleotides embedded in the support in the basic medium,
(iii) concentrating the reaction mixture obtained in step (ii) in a vacuum concentrator.
(iv) dissolving the solid residue obtained in step (iii) in water and subjecting to gel filtration to remove low molecular weight impurities.
(v) concentrating the eluent obtained in step (iv) under vacuum concentrator and subjecting to high performance liquid chromatography (HPLC) to obtain the free fully deprotected oligodeoxyribo- and oligoribonucleotides.
The 2'-deoxyribonucleosidephosphoramidites and ribonucleosidephosphoramidites used for the synthesis of oligodeoxyribo- and ribonucleotides in step (i) carrying conventional protecting groups may be selected from benzoyl for adenine, benzoyl or acetyl for cytosine and isobutyryl for guanine or labile protecting groups such as phenoxyacetyl for adenine and guanine and isobutyryl or acetyl for cytosine; dimethylformamidine (DMF) for adenine, guanine and cytosine; p-tert-butylphenoxyacetyl- for adenine, guanine and cytosine and the like for exocyclic amino functions of nucleic bases.
The basic medium employed in step (ii) may be selected from aq. ammonia, aq. methylamine, aq. ammonia-methylamine (1:1, v/v) mixture and the like.
The temperature required for cleaving synthesized oligomers from the support and simultaneous removal of protecting groups may be selected from the range 30.degree.-65.degree. C. depending upon the protecting groups employed for nucleic bases.
The time required for cleaving synthesized oligomers from the support and simultaneous removal of protecting groups may be selected from the range 5-120 min depending upon the protecting groups employed for nucleic bases.